Calibrator/control for simultaneous assay of proteins capable of complexing with one another

ABSTRACT

Disclosed herein are compositions and methods comprising two or more proteins in which at least one of the proteins has been altered to reduce their mutual recognition and binding. Such compositions are useful as reference, calibrators or controls in methods and assays for determining the amount of one or more of the proteins that may be present in a sample of interest or in confirming the presence of one or more of the proteins in the sample. More particularly, it relates to compositions and methods comprising altered placental growth factor-1 (PlGF-1) and soluble fms-like tyrosine kinase (sFlt-1) and methods for determining the amount or confirming the presence of sFlt-1 and/or PlGF-1 in a sample of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/349,695, filed Jan. 7, 2009, now U.S. Pat. No. 8,148,157 which claims the benefit of U.S. Provisional Application No. 61/019,443, filed Jan. 7, 2008.

FIELD OF THE INVENTION

The present invention relates to compositions and methods comprising two or more proteins altered to prevent their mutual recognition and binding. The compositions can be used as reference, calibrator or control in analytical assays capable of detecting both altered and unaltered or native forms of one or more of the proteins.

BACKGROUND OF THE INVENTION

Pre-eclampsia is a syndrome of hypertension, edema, and proteinuria that affects 5 to 10% of pregnancies and results in substantial maternal and fetal morbidity and mortality. Pre-eclampsia accounts for at least 200,000 maternal deaths worldwide per year. The symptoms of pre-eclampsia typically appear after the 20^(th) week of pregnancy.

Development of a fetus and placenta is mediated by several growth factors. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen, and angiogenic inducer. VEGF mediates vascular permeability and has been shown to be involved in glomerular capillary repair. VEGF binds as a homodimer to one of two homologous membrane-spanning receptor tyrosine kinases, the fms-like tyrosine kinase (Flt-1) and the kinase domain receptor (KDR).

Placental growth factor (PlGF) is a VEGF family member that is also involved in placental development. PlGF is expressed by cytotrophoblasts and syncytiotrophoblasts and is capable of inducing proliferation, migration, and activation of endothelial cells. PlGF binds as a homodimer to the Flt-1 receptor, but not the KDR receptor. Both PlGF and VEGF contribute to the mitogenic activity and angiogenesis that are critical for the developing placenta.

A soluble form of the Flt-1 receptor (sFlt-1) has been identified in a cultured medium of human umbilical vein endothelial cells and in vivo expression was subsequently demonstrated in placental tissue. sFlt-1 is a splice variant of the Flt-1 receptor which lacks the transmembrane and cytoplasmic domains. sFlt-1 binds to VEGF with high affinity but does not stimulate mitogenesis of endothelial cells. sFlt-1 is believed to act as a “physiologic sink” to down-regulate VEGF signaling pathways. Regulation of sFlt-1 levels therefore works to modulate VEGF and VEGF signaling pathways. Careful regulation of VEGF and PlGF signaling pathways is critical for maintaining appropriate proliferation, migration, and angiogenesis by trophoblast cells in the developing placenta.

A single gene codes for human PlGF. However, splicing of the mature PlGF mRNA results in three different length isoforms: PlGF-1 (PlGF131), PlGF-2 (PlGF152), and PlGF-3 (PlGF203). Another variant, PlGF-4, has been reported (Yang, et al, J Reprod Immunol, v 60, p 53-60, 2003). PlGF is secreted as a glycosylated homodimer.

Recently it has been shown that sFlt-1 and PlGF may be used individually or in combination as biomarkers to predict, diagnose, or monitor pre-eclampsia (Levine et al, NEJM, v 350, p 672-683, 2004).

The amino acid sequence of mature human PlGF-1, amino acid residues 1-132, has been published and is available from the Protein Data Bank identified as PDB 1FZV (Iyer, et al, J. Biol Chem, v 276, p 12153-12161, 2001). This sequence is identified herein as SEQ ID NO:1:

MLPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALER LVDVVSEYPS EVEHMFSPSC VSLLRCTGCC GDENLHCVPV ETANVTMQLL KIRSGDRPSY VELTFSQHVR CECRPLREKM KPERCGDAVP RR

Diagnosis of an individual at risk for, or having pre-eclampsia may be made by determining the presence or amount of vascular endothelial growth factor, particularly PlGF, and/or receptor tyrosine kinase, particularly, sFlt-1 in a biological sample (such as urine, whole blood, serum, plasma, saliva, and so forth) taken from the individual. In analytical assays reference, calibrator and control compositions are essential for purposes of determining the amount or confirming the presence of a target analyte and, for establishing accuracy and precision of the analytical assay. The preparation of such compositions in liquid or dry form usually doesn't present difficulties if the analyte is readily available, soluble in an appropriate solvent—usually aqueous for biological analytes, stable, and does not interact deleteriously with other components that may be present in the composition. As noted above, PlGF binds sFlt-1 to form a stable association complex. As a result compositions comprising the native proteins together in independent amounts suitable for use as a reference, calibrator or control in analytical assays to detect PlGF or sFlt-1 or both PlGF and sFlt-1 cannot be prepared. Although compositions comprising the individual, separated proteins may be prepared it would be advantageous to be able to prepare compositions comprising both proteins together. Thus, a need exists for reference, calibrator or control compositions comprising these proteins together in known and independent amounts. This need has been met with the present invention.

SUMMARY OF THE INVENTION

In one aspect the present invention relates to a composition comprising two or more proteins, one or more of the proteins having been altered to sufficiently reduce or substantially prevent or eliminate mutual recognition and binding. Such a composition is useful as a reference, calibrator or control in analytical assays for one or more of the proteins in the composition.

Considering for clarity two proteins unaltered/native proteins A and B which form a non-covalent association complex, the term “substantially prevent or eliminate their mutual recognition and binding” means that in an assay to determine their mutual binding, binding of altered A to unaltered/native B, or binding of unaltered/native A to altered B, or binding of altered A to altered B is not detectable, or barely detectable, or the mutual affinity as measured quantitatively by determination of affinity constants is less than approximately 10% of that observed for unaltered/native A and unaltered/native B. The term “sufficiently reduce” means that mutual binding occurs, but it has been reduced to a degree that is acceptable for a particular application.

Consider a case where the presence or amount of unaltered or native protein A is to be determined in an analytical assay, which assay utilizes one or more receptors specific for epitopes of protein A. And consider that protein A has been altered to reduce or substantially eliminate binding to protein B. Although protein A has been altered the epitopes remain intact or acceptably intact such that they retain their ability to recognize and bind the receptors. Thereby, altered protein A is acceptable for use in calibrating the assay, confirming the presence of unaltered/native protein A in a sample, or for verifying the accuracy and precision of the assay for unaltered/native protein A. Thus, in general, receptors are capable of recognizing and binding both altered and unaltered/native forms of a protein. Analytical assays comprising receptors are usually immunoassays, which assays employ as receptors polyclonal or monoclonal antibodies, whole, polymeric and/or chimeric forms of antibodies or antibody fragments. Other kinds of receptors are also used, such as aptamers (U.S. Pat. No. 5,840,867; U.S. Pat. No. 6,207,388). In an analytical assay for the determination of unaltered or native protein B using receptors specific for epitopes of protein B, it is not necessary that epitopes of altered protein A remain intact. It is only important that mutual recognition and binding of altered protein A and protein B have been sufficiently reduced or substantially eliminated.

If both protein A and protein B have been altered to reduce or substantially eliminate their mutual recognition and binding then in an analytical assay for determination of unaltered/native protein A or an analytical assay for determining unaltered/native protein B or an analytical assay for determination of both unaltered/native proteins A and B—which assays utilize receptors specific for epitopes of protein A and receptors specific for epitopes of protein B, these epitopes in the altered proteins retain the ability to recognize and bind the receptors used in the assay.

Compositions comprising both altered protein A and altered protein B together can then be used for calibrating the assays, confirming the presence of unaltered/native protein A, or unaltered/native protein B, or both unaltered/native protein A and unaltered/native protein B, and for verifying accuracy and precision of the assays.

In another aspect the present invention relates to a reference, calibrator or control composition for use in an assay for a first protein or a second protein or both first and second proteins, wherein one or more amino acids or one or more non-amino acid groups of the first protein or the second protein or the first protein and the second protein have been deleted, modified, or replaced with a different amino acid or non-amino acid group or groups thereby reducing or substantially eliminating mutual binding of the first protein and the second protein.

In another aspect the present invention relates to a composition comprising a receptor tyrosine kinase, preferably fms-like tyrosine kinase, more preferably sFlt-1 and a vascular endothelial growth factor either or both altered, by amino acid or glycosyl deletion, modification or replacement. The vascular endothelial growth factor may be a placental growth factor and preferably, PlGF-1. A preferred composition comprises sFlt-1 and altered PlGF-1 having alanine in place of:

-   -   a) proline at position 25 of SEQ ID NO:1, or     -   b) glutamine at position 27 of SEQ ID NO:1, or     -   c) cysteine at position 60 of SEQ ID NO:1, or     -   d) aspartate at position 72 of SEQ ID NO:1 or     -   e) glutamate at position 73 of SEQ ID NO:1, or     -   f) asparagine at position 84 of SEQ ID NO:1, or     -   g) proline at position 98 of SEQ ID NO:1, or     -   h) tyrosine at position 100 of SEQ ID NO:1, or         altered PlGF-1 having glycine in place of cysteine at position         70 of SEQ ID NO:1, or any combination of the alanine         replacements in a) to h) and the glycine replacement.

A preferred composition comprises sFlt-1 and altered PlGF-1 having alanine in place of aspartate at position 72 of SEQ ID NO:1 and alanine in place of glutamate at position 73 of SEQ ID NO:1.

Another preferred composition comprises sFlt-1 and altered PlGF-1 having glycine in place of cysteine at position 70 of SEQ ID NO:1, alanine in place of aspartate at position 72 of SEQ ID NO:1, and alanine in place of glutamate at position 73 of SEQ ID NO:1.

In another aspect the present invention relates to a method for calibrating an assay for a protein in a sample comprising the steps of:

-   -   1) contacting a composition as described above comprising known         amounts of the protein with a receptor specific for a first         epitope of the protein, thereby forming a complex comprising the         receptor and the protein of the composition;     -   2) contacting the complex formed in step 1) with a labeled         receptor specific for a second epitope of the protein, thereby         forming a complex comprising receptor, the protein of the         composition, and labeled receptor;     -   3) detecting a signal from bound labeled receptor or a signal         from free labeled receptor; and,     -   4) associating the signal from free or bound labeled receptor         with the known amounts of the protein in the composition.     -   In another aspect the invention relates to a method for         calibrating an assay for a protein in a sample comprising the         steps of:     -   1) contacting a composition as described above comprising known         amounts of the protein with an immobilized receptor specific for         a first epitope of the protein, thereby forming a complex         comprising immobilized receptor and the protein of the         composition;     -   2) contacting the complex formed in step 1) with a labeled         receptor specific for a second epitope of the protein, thereby         forming a complex comprising immobilized receptor, the protein         of the composition and labeled receptor;     -   3) separating bound labeled receptor from free labeled receptor;     -   4) detecting a signal from bound labeled receptor or a signal         from free labeled receptor; and,     -   5) associating the signal from free or bound labeled receptor         with the known amounts of the protein in the composition.

In yet another aspect the present invention relates to a method for calibrating an assay for a receptor tyrosine kinase and/or a vascular endothelial growth factor in a sample comprising the steps of:

-   -   1) preparing a composition comprising a known or pre-determined         amount of the receptor tyrosine kinase and a known or         pre-determined amount of the vascular endothelial growth factor         either or both altered as described above to reduce or         substantially eliminate their mutual recognition and binding;     -   2) contacting the composition with a receptor specific for a         first epitope of the receptor tyrosine kinase and/or a receptor         specific for a first epitope of the vascular endothelial growth         factor, thereby forming first complexes of receptor specific for         the first epitope of the receptor tyrosine kinase and receptor         tyrosine kinase and/or receptor specific for the first epitope         of the vascular endothelial growth factor and endothelial growth         factor;     -   3) contacting complexes formed in step 2) with a labeled         receptor specific for a second epitope of the receptor tyrosine         kinase and/or a labeled receptor specific for a second epitope         of the endothelial growth factor; thereby forming second         complexes comprising receptor specific for the first epitope of         receptor tyrosine kinase, receptor tyrosine kinase and labeled         receptor specific for the second epitope of the receptor         tyrosine kinase and/or receptor specific for the first epitope         of endothelial growth factor, endothelial growth factor and         labeled receptor specific for the second epitope of endothelial         growth factor;     -   4) separating bound labeled receptor specific for receptor         tyrosine kinase from free labeled receptor specific for receptor         tyrosine kinase and/or bound labeled receptor specific for         endothelial growth factor from free labeled receptor specific         for endothelial growth factor;     -   5) detecting a signal from bound labeled receptor specific for         receptor tyrosine kinase or a signal from free labeled receptor         specific for receptor tyrosine kinase; and/or,     -   6) detecting a signal from bound labeled receptor specific for         endothelial growth factor or a signal from with free labeled         receptor specific for endothelial growth factor; and,     -   7) associating the signal from free or bound labeled receptor         specific for receptor tyrosine kinase and/or the signal from         free or bound labeled receptor specific for endothelial growth         factor with the known amounts of receptor tyrosine kinase and/or         endothelial growth factor in the composition.

In a preferred embodiment the receptor tyrosine kinase is sFlt-1 and the endothelial growth factor is PlGF-1, which PlGF-1 has been altered to have alanine in place of:

-   -   a) proline at position 25 of SEQ ID NO:1, or     -   b) glutamine at position 27 of SEQ ID NO:1, or     -   c) cysteine at position 60 of SEQ ID NO:1, or     -   d) aspartate at position 72 of SEQ ID NO:1 or     -   e) glutamate at position 73 of SEQ ID NO:1, or     -   f) asparagine at position 84 of SEQ ID NO:1, or     -   g) proline at position 98 of SEQ ID NO:1, or     -   h) tyrosine at position 100 of SEQ ID NO:1, or         altered PlGF-1 having glycine in place of cysteine at position         70 of SEQ ID NO:1, or any combination of the alanine         replacements in a) to h) and the glycine replacement.

In a more preferred embodiment the receptor tyrosine kinase is sFlt-1 and the endothelial growth factor is PlGF-1, which PlGF-1 has been altered to have alanine in place of aspartate at position 72 of SEQ ID NO:1 and alanine in place of glutamate at position 73 of SEQ ID NO:1.

In another more preferred embodiment, the receptor tyrosine kinase is sFlt-1 and the endothelial growth factor is PlGF-1, which PlGF-1 has been altered to have glycine in place of cysteine at position 70 of SEQ ID NO:1, alanine in place of aspartate at position 72 of SEQ ID NO:1, and alanine in place of glutamate at position 73 of SEQ ID NO:1.

In yet another aspect the present invention relates to a method for determining the amount or confirming the presence of a protein in a sample comprising the steps of:

-   -   a) contacting the sample with immobilized receptor specific for         a first epitope of the protein, thereby forming a first complex         comprising immobilized receptor specific for the first epitope         of the protein and the protein;     -   b) contacting the first complex with labeled receptor specific         for a second epitope of the protein, thereby forming a second         complex of receptor specific for the first epitope of the         protein, the protein, and labeled receptor specific for the         second epitope of the protein;     -   c) separating labeled receptor that is bound in the second         complex from free labeled receptor;     -   d) determining a signal from labeled receptor that is bound in         the second complex or a signal from free labeled receptor;     -   e) contacting immobilized receptor specific for the first         epitope of the protein with a composition as described above         comprising a known or pre-determined amount of the protein,         thereby forming a third complex comprising immobilized receptor         specific for the first epitope of the protein and the protein of         the composition;     -   f) contacting the third complex with labeled receptor specific         for the second epitope of the protein, thereby forming a fourth         complex of receptor specific for the first epitope of the         protein, the protein of the composition, and labeled receptor         specific for the second epitope of the protein;     -   g) separating labeled receptor that is bound in the fourth         complex from free labeled receptor;     -   h) determining a signal from labeled receptor that is bound in         the fourth complex or a signal from free labeled receptor; and,     -   i) comparing the signals determined in d) and h) as confirmation         of the presence of the protein or as a measure of the amount of         the protein in the sample.

In a preferred embodiment the protein to be determined is a vascular endothelial growth factor. In a more preferred embodiment the protein to be determined is PlGF and particularly PlGF-1 and the second protein in the composition is a receptor tyrosine kinase. In a more preferred embodiment the protein to be determined is PlGF-1 and the second protein in the composition is sFlt-1. In a more preferred embodiment the protein to be determined is PlGF-1, the second protein in the composition is sFLt-1 and PlGF-1 of the composition has been altered to comprise alanine in place of:

-   -   a) proline at position 25 of SEQ ID NO:1, or     -   b) glutamine at position 27 of SEQ ID NO:1, or     -   c) cysteine at position 60 of SEQ ID NO:1, or     -   d) aspartate at position 72 of SEQ ID NO:1 or     -   e) glutamate at position 73 of SEQ ID NO:1, or     -   f) asparagine at position 84 of SEQ ID NO:1, or     -   g) proline at position 98 of SEQ ID NO:1, or     -   h) tyrosine at position 100 of SEQ ID NO:1, or         altered PlGF-1 having glycine in place of cysteine at position         70 of SEQ ID NO:1, or any combination of the alanine         replacements in a) to h) and the glycine replacement.

In a more preferred embodiment the protein to be determined is PlGF-1, the second protein of the composition is sFlt-1 and PlGF-1 of the composition comprises alanine in place of aspartate at position 72 of SEQ ID NO:1 and alanine in place of glutamate at position 73 of SEQ ID NO:1.

In another more preferred embodiment, the protein to be determined is PlGF-1, the second protein of the composition is sFlt-1 and PlGF-1 of the composition comprises glycine in place of cysteine at position 70 of SEQ ID NO:1, alanine in place of aspartate at position 72 of SEQ ID NO:1, and alanine in place of glutamate at position 73 of SEQ ID NO:1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates ELISA-based binding of PlGF-1 variants to the soluble portion of Flt-1. Binding of PlGF-1 variants to human Flt-1, coated at 0.5 μg/ml on a 96-well plate, was performed using increasing concentrations of soluble proteins ranging between 1 and 16 ng/ml. Wild type PlGF-1 was used as a positive control.

FIG. 1B illustrates ELISA-based binding of PlGF-1 variants to the soluble portion of Flt-1. Percentage of binding of PlGF-1 variants at a concentration of 8 ng/ml calculated with respect to the binding of wt PlGF-1. The results shown represent the average of three independent experiments.

FIG. 2 illustrates the purity of three PlGF recombinant proteins. Silver stain (A: lanes 1, 2 and 3) and Western blot by monoclonal Rat-4 (B: lanes 4, 5 and 6). Lane assignment: M:SeaBlue ladder (Invitrogen), lane 1,4—P126(DE), lane 2,5—P126 (AA) and lane 3,6—P126(GAA).

DETAILED DESCRIPTION

Although the present invention will be described in terms of certain preferred embodiments relating to pre-eclampsia and biomarker proteins sFlt-1 and PlGF-1, it should be understood that the invention relates to any protein composition and its use in which one or more component proteins of the composition have been altered to reduce their mutual recognition and binding.

Whether the pre-eclampsia biomarker proteins are determined using a single assay platform or a single kit, or determined separately in independent assays or kits, it is advantageous to have a control or calibrator comprising both biomarker proteins together in the same formulation having known or pre-determined concentrations and desired concentration ratios. There are at least two problems associated with using sFlt-1 and PlGF together in native or unaltered form to prepare reference, calibrator or control compositions: firstly, sFlt-1 and PlGF bind to each other through a specific binding domain present on each protein, as already noted, and secondly, in the serum of mid- to late-term pregnant women, sFlt-1 is typically present at a significant excess relative to PlGF whether or not they are afflicted with pre-eclampsia. Unmodified or native PlGF combined and stored together with unmodified or native sFlt-1 will not serve satisfactorily in a composition used to calibrate an assay for detection of PlGF or sFlt-1 because of the nearly quantitative binding of PlGF to sFlt-1.

Amino acid changes have been made to PlGF that reduce or substantially eliminate mutual recognition and binding of sFlt-1 and PlGF (Errico et al J. Biol. Chem. 279, 43929-43939, 2004). These amino acid changes do not have a significant impact on the overall protein structure of PlGF. Binding epitopes remain intact and permit these altered proteins to be combined and stored together with sFlt-1 in a composition for use as a reference, calibrator or control for assays designed to detect unaltered or native PlGF, unaltered or native sFlt-1, or both.

Targeting amino acid modifications, deletions or replacements to PlGF in order to reduce or substantially eliminate binding to sFlt-1 has been facilitated because the amino acid sequence of PlGF and 3-D crystal structure are available.

In general it would be advantageous to know secondary, tertiary, and quaternary structures, post-translational modifications (eg phosphorylation, glycosylation, sulfation, and ubiquitination), 3-D crystal structures of binding proteins and 3-D crystal structures of the proteins engaged in their association complex. However, this information is not required in order to practice the present invention. Although modification, deletion or replacement of groups associated with post-translational modifications can be carried out, modification, deletion or replacement of one or more amino acids of one or more of the proteins that engage in mutual recognition and binding is preferred. Site-specific chemical modification of proteins is well known in the art (Techniques in Protein Modification, Lundblad R L, CRC Press, 1995; Chemical Reagents for Protein Modification, Lundblad, R L, CRC Press, 3^(rd) Ed, 2005). Chemical/synthetic modification of amino acids can be used to practice the present invention. A preferred approach involves genetic engineering techniques. Obtaining the amino acid sequence of a protein directly is standard practice in the art. Similarly, it is standard practice in the art to obtain the amino acid sequence of a protein indirectly from the nucleotide sequence of the gene that codes for the protein. The nucleotide sequence of a gene can be readily obtained. And, when the gene is available site-directed mutagenesis can be carried out to delete, replace, or modify one or more amino acids. This can be done in a random manner or in a predetermined manner. A protein that is altered or mutated using site-directed mutagenesis can be cloned and made readily available. Protein and genetic engineering details and protocols are readily available from numerous publications and citations therein (Molecular Cloning, Sambrook J and Russell D W, Cold Spring Harbor Laboratory Press, 2002; Recombinant Gene Expression Protocols, Tuan R S ed, Humana Press, 1997; Methods in Molecular Biology and Protein Chemistry, Spangler B D, John Wiley & Sons Ltd. 2002; Genetic Engineering Fundamentals, Kammermeyer K and Clark V L, Marcel Dekker Inc, 1989; Mayo et al, Nature v 306, p 86-88, 1983; Suggs et al, Proc Nat Acad Sci USA v 78, p 6613-6617 1981; Scott et al Nature v 302, p 538-540, 1983; Helfman et al, Proc Nat Acad Sci USA, v 80, p 31-35, 1983; Young et al, Proc Nat Acad Sci USA, v 80, p 1194-1198, 1983; U.S. Pat. No. 4,237,224; U.S. Pat. No. 4,273,875; U.S. Pat. No. 4,293,652; U.S. Pat. No. 4,870,009).

The altered protein can be tested to determine if mutual recognition and binding with its partner protein(s) have been reduced or substantially eliminated. This can be carried out using experimental protocols well known in the art. The altered protein also can be tested to determine if epitopes have been sufficiently undisturbed compared with unaltered or native protein using epitope specific receptors/antibodies. Affinity can be characterized quantitatively or qualitatively. (Errico et al, J Biol Chem, v 279, p 43929-43939, 2004; Piehler et al, J Immunol Methods, v 201(2), p 189-206, 1997; Casasnovas et al, v 270, p 13216-13224, 1995; Boone et al, J Virol, v 11, p 515-519, 1973; U.S. Pat. No. 7,081,346; U.S. Pat. No. 5,324,633; U.S. Pat. No. 4,340,668; US 2005/0175999).

Whatever the nature of the group or groups (amino acids and/or non-amino acids) altered, or the nature of the protein alteration—modification (direct chemical modification—oxidation, reduction, etc), deletion or replacement of the group(s), or whether one or each of the proteins that participate in mutual recognition and binding are altered, the two important functional features are: 1) mutual recognition and binding of an altered protein to an unaltered partner protein or binding of partner proteins when each have been altered is such that mutual recognition and binding is sufficiently reduced or substantially eliminated and 2) one or more epitopes of any altered protein retain binding properties sufficiently similar or substantially identical to the epitope(s) in the unaltered or native protein if this property is required for the particular application as discussed earlier.

Example I

Human PlGF-1 and variants, sFlt-1, anti-human PlGF-1 antibodies directed to human PlGF, binding characteristics of PlGF and variants to sFlt-1, ELISA assay for determining PlGF and other materials and experimental protocols have been described by Errico et al, J Biol Chem, v 279, p 43929-43939, 2004, and are reproduced herein in part. The Errico et al reference can be consulted for details regarding cell cultures, plasmids, selection of cell lines, and other materials and experimental protocols not explicitly provided herein.

Materials

As described by Errico et al., anti-human PlGF monoclonal antibodies and human Flt-1 (Flt-1/Fc chimera) are available from R&D Systems (Minneapolis, Minn. USA). Goat anti-mouse IgG-horseradish peroxidase (HRP) is available from Santa Cruz Biotechnology (Santa Cruz, Calif. USA; www.scbt.com).

Construction of PlGF Variants

Errico et al. obtained PlGF variants using PCR techniques carried out using the plasmid named pchPlGF-1 as template and PCR was performed using complementary primers mapping the region encoding the amino acid to be mutated to alanine and bearing the specific nucleotide modification. For the preparation of the PlGF variant having the double mutation, primers carrying both mutations were utilized. Amplified DNA was purified and used to transform competent bacteria. The plasmids were sequenced in both directions using the dideoxynucleotide method. The following PlGF-1 single residues were mutated to Ala: Asn-16, Pro-25, Gln-27, Cys-60, Asp-72, Glu-73, Asn-74, Asn-84, Pro-98, and Tyr-100. The double mutant Asp 72 to Ala and Glu 73 to Ala of PlGF-1 was also generated.

Calibrator/Control Compositions Comprising Altered PlGF-1

Calibrators/controls comprising altered PlGF-1 and sFlt-1 are prepared by combining unaltered sFlt-1 with an altered PlGF-1, in particular, the double mutant in which alanines replace aspartate at position 72 of SEQ ID NO:1 and glutamate at position 73 of SEQ ID NO:1 or the triple mutant in which there is an additional mutation of glycine replacing cysteine at position 70. These may be combined individually from dry form preparations or from working aqueous stock solutions prepared using any suitable buffer at a desired pH (such as, phosphate in saline (PBS), pH 7.5) comprising any other addenda that may be useful or required—such as anti-oxidants, preservatives, etc. For illustrative purposes, the concentration of altered PlGF-1 is in the range of 0 to about 1000 pg/mL, and sFlt-1 fixed at 100 pg/mL but other concentration ranges for both may be used. The unaltered sFlt-1 is combined with altered PlGF of the double mutant or the triple mutant in PBS (10 g NaCl, 0.25 g KCl, 1.8 g Na₂HPO₄, 0.3 g KH₂PO₄, pH 7.5) to produce the following set of reference, calibrator or control materials:

Altered PlGF1 (pg/mL) SFlt-1 (pg/mL) 0 100 50 100 100 100 500 100 1000 100 ELISA For PlGF

The quantity of PlGF in a sample of serum obtained from a pregnant woman is determined using an ELISA for PlGF. The ELISA (described in detail below) is calibrated using the set of solutions comprising altered PlGF-1 and sFlt-1 described above. The signal observed for each PlGF-1 level of the set is associated with the concentration of altered PlGF-1. The association can be represented in graphic form or correlated using appropriate statistical and mathematical calibration methods. The signal observed in the ELISA assay using the serum sample is compared with the calibration graph to determine the concentration of PlGF in the sample or transformed into concentration units using the established mathematical association.

The ELISA is carried out as follows: for determination of PlGF in a sample, one anti-human PlGF-1 monoclonal antibody at 1 μg/ml in PBS is used to coat a 96-well plate at 100 μl/well and incubated overnight at 4° C. The wells are washed once with PBS containing 0.05% TWEEN 20 (PBT) and non-specific binding sites are blocked by introducing 1% bovine serum albumin in PBS at 280 μl/well and incubation for 3 h at room temperature (RT). The wells are aspirated and kept in the cold until use. During the assay, 100 μl of each calibrator level or serum sample is appropriately diluted in PBET (PBS containing 0.1% bovine serum albumin, 5 mM EDTA, 0.05% Tween 20) and incubated for 1 hour at 37° C. The wells are washed five times by PBT and another anti-human PlGF-1 monoclonal antibody (this one HRP conjugated) diluted in PBET at 37 ng/ml, is added to the wells and incubated for 1 h at 37° C. The wells are washed five times with PBT and 100 μl of HRP substrate composed of 1 mg/ml of orthophenylenediamine in 50 mM citrate phosphate buffer, pH 5 and 0.006% H₂O₂ is added and incubated for 30 min in the dark at RT. The reaction is stopped by adding 25 μl/well of 4 N H₂SO₄, and the signal absorbance is measured at 490 nm on a microplate reader.

Comparison of Altered PlGF-1 and Unaltered PlGF Binding to sFlt-1

Errico et al. has described the experiment to determine the binding of altered PlGF-1 and unaltered/native PlGF-1 to Flt-1. Basically, a 96-well plate is coated with a soluble human Flt-1 (Flt-1/Fc chimera) at 0.5 μg/ml in PBS, pH 7.5, 100 μl/well, overnight at RT. The plate is washed five times with PBT, and after the blocking non-specific sites of wells with bovine serum albumin solution as described above, the binding reaction is allowed to proceed by adding altered PlGF-1 or unaltered/native PlGF to a well and incubating for 1 h at 37° C. and 1 h at RT. The wells are washed with PBT as described above and incubated with a biotinylated anti-human PlGF-1 polyclonal antibody, 300 ng/ml in PBET, for 1 h at 37° C. and 1 h at RT. Detection is performed as described above in the ELISA assay and the signals obtained with altered PlGF-1 and unaltered/native PlGF-1 are compared. The results obtained by Errico et al. are reproduced in FIG. 1A and FIG. 1B.

Example II Comparing Recombinant PlGF (DE) and PlGF (AA)

Experiment Purpose:

Two recombinant PlGF proteins were evaluated (1) for their binding reactivity to monoclonal antibody specific to human PlGF, and (2) for their binding reactivity to sFlt, the formation of ligand:receptor complex.

Materials and Reagents:

(1) Recombinant PlGF:

Two versions of purified recombinant PlGF were used. The protein consists of a 21-amino-acid leader sequence that does not belong to PlGF. The leader sequence contains a “6×His” tag and a 4-amino-acid Xa recognition and cleavage site.

SEQ ID NO: 2: Leader sequence: MRGS HHHHHH GSGSGSG IEGR The PlGF portion sequence in PlGF (DE): Amino acid sequence corresponds to wild-type PlGF amino acids 4-132 of SEQ ID NO:1, resulting in the following DE amino acid sequence:

SEQ ID NO: 3: AVPPQQWALS AGNGSSEVEV VPFQEVWGRS YCRALERLVD VVSEYPSEVE HMFSPSCVSL LRCTGCCG DE  NLHCVPVETA NVTMQLLKIR SGDRPSYVEL TFSQHVRCEC RPLREKMKPE RCGDAVPRR The PlGF portion sequence in PlGF (AA): Amino acid sequence corresponds to wild-type PlGF amino acids 4-132 of SEQ ID NO:1 with two mutations made at amino acid positions 72 and 73 in SEQ ID NO:1, resulting in the following AA amino acid sequence:

SEQ ID NO:4 : AVPPQQWALS AGNGSSEVEV VPFQEVWGRS YCRALERLVD VVSEYPSEVE HMFSPSCVSL LRCTGCCG AA  NLHCVPVETA NVTMQLLKIR SGDRPSYVEL TFSQHVRCEC RPLREKMKPE RCGDAVPRR (2) Recombinant sFlt:

Full length sFlt was obtained from Scios Inc. (Mountain View, Calif. USA; www.sciosinc.com) (Lot #9225-89), consists of 687 amino acids of soluble fms-like tyrosine kinase 1 (sFlt-1).

sFlt-1 sequence:

SEQ ID NO: 5: MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWS LPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAI YIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIW DSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTL VLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYT CRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPEVVWLKD GLPATEKSARYLTRGYSLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKA VSSFPDPALYPLGSRQILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADS NMGNRIESITQRMAIIEGKNKMASTLVVADSRISGIYICIASNKVGTVGRNISFYITDVPNG FHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSISKQKMAITKEHSITLN LTIMNVSLQDSGTYACRARNVYTGEEILQKKEITIRGEHCNKKAVFSRISKFKSTRNDCTTQ SNVKH (3) Monoclonal Antibody to Human sFlt-1 and to Human PlGF:

monoclonal Ab ID Source Cat # Lot Clone RD-1 mouse anti-sFlt RD Sys CGG07605B 49560 RD-2 mouse anti-sFlt RD Sys BYC01605A 49543 Rat-1 rat anti-PlGF RD Sys n/a 1103925 358903 Rat-2 rat anti-PlGF RD Sys n/a 1103933 358939 Rat-3 rat anti-PlGF RD Sys n/a 1103931 358932 Rat-4 rat anti-PlGF RD Sys n/a 1103926 358905 Rat-5 rat anti-PlGF RD Sys n/a 1103927 358907 MS-1 mouse anti-PlGF RD Sys MAB264 n/a 37203

Experiment Examples

(1) ELISA assay-1:

-   -   High-binding microtiter plate was coated with recombinant         PlGF(DE) or PlGF(AA) at 0.5 ug/mL and blocked with BSA/PBS     -   Standard ELISA procedure consists of monoclonal antibody         dilution in casein/PBS; dilution of HRP conjugated donkey         anti-mouse IgG or donkey anti-rat IgG at 1:3 K in casein/PBS;         100 uL/well sample or conjugate volume; each step incubation at         37 C/30 min/shake; 6 times plate washing, 100 uL OPD substrate         development for 25 C/30 min; 25 uL stop solution; record OD at         492 nm.     -   ELISA results assay 1 are shown in Table 1.

TABLE 1 Recognition of monoclonal anti-PlGF to recombinant PlGF (unaltered and altered) Antibody binding activity to coated PlGF (OD) Monoclonal anti-PlGF ID, clone # and Coated concentration (ng/mL) recombinant Ms-1 Rat 1 Rat 2 Rat 4 Rat 3 Rat 5 PlGF Ab (10 ng/mL) Ab (100 ng/mL) (0.5 μg/mL) 37203 358903 358939 358905 358932 358907 PlGF-1 (DE) 1.823 2.098 2.237 2.114 1.233 1.256 PlGF-1 (AA) 2.650 1.789 2.233 1.935 1.305 1.297

-   -   Conclusion: All monoclonal antibodies tested reacted to both         PlGF(DE) and PlGF(AA), indicating that D72/E73A mutation did not         affect monoclonal antibody binding and these antibody epitope         locations were not at these two mutation sites.         (2) ELISA Assay-2:     -   High-binding microtiter plate was coated with recombinant         PlGF(DE) or PlGF(AA) at 0.5 ug/mL and blocked with BSA/PBS     -   Standard ELISA procedure consists of 1st plate incubation with         diluted sFlt in casein/PBS at various concentrations; 2nd plate         incubation with mixed anti-sFlt solution comprising two         monoclonal antibodies of RD-1 and RD-2 each at 0.1 μg/mL; 3rd         plate incubation with HRP conjugated donkey anti-mouse at IgG at         1:4 K dilution in casein/PBS; and 4th plate incubation with 100         μL OPD substrate development for 30 min at 25° C. 1st, 2nd and         3rd plate incubation steps are for 15-20 min/shake at 37° C.; 6         times plate washing between each step. 25 μL stop solution after         4th incubation and record OD at 492 nm.     -   ELISA results assay-2 are shown in Table 2.

TABLE 2 Binding of sFlt to recombinant PlGF (unaltered and altered) Complex formation of Coated sFlt to coated PlGF recombinant sFlt concentration PlGF (ng/mL) at incubation (0.5 μg/mL) 1800 600 200 66.67 BSA (control) 0.004 0.006 0.010 0.020 PlGF-1 (DE) 1.728 1.204 0.524 0.316 PlGF-1 (AA) 0.150 0.080 0.060 0.040 * Bound sFlt:PlGF complex were detected by mouse anti-sFlt and HRP anti-mouse conjugate

-   -   Conclusion: sFlt formed receptor:ligand complex with coated         PlGF(DE). However, such complex formation was greatly reduced         with PlGF(AA) mutant, indicating that amino acid positions 72         and 73 in SEQ ID NO:1 were critical for sFlt-1 binding and         complex formation.         (3) Biacore Assay:     -   sFlt were immobilized on Biacore chip FC-2 via NHS/EDC coupling         to a RU=6738. FC-1 was blank as negative control.     -   PlGF(DE) or PlGF(AA) were injected to FC-1 and FC-2 to evaluate         complex formation     -   Biacore results are shown in Table 3.

TABLE 3 Biacore measurement of sFlt:PlGF complex recomb hu PlGF 20 μg/mL CM5 Chip: PlGF (DE) PlGF (AA) Bicore FC1: blank 11 12 (RU) FC2: sFlt 156 30 FC2 − FC1 145 18

-   -   Conclusion: Injected PlGF(DE) bound to immobilized sFlt to form         receptor:ligand complex while injected PlGF(AA) bound to         immobilized sFlt poorly.

Example III

Comparison between Wild-type Recombinant PlGF and Two Additional PlGF Mutants

Experiment Purpose:

Three additional recombinant PlGF proteins were constructed and evaluated (1) for their binding reactivity to monoclonal antibody specific to human PlGF, and (2) for their binding reactivity to sFlt, the formation of ligand:receptor complex.

Materials and Reagents:

(1) Three Additional Recombinant PlGF Proteins were Constructed as Follows:

P126(DE): recombinant P1GF wild type: SEQ ID NO: 6: MRGSAVPPQQWALSAGNGSSEVEVVPFQEVWGRSYCRALERLVDVVSEYP SEVEHMFSPSCVSLLRCTGCCGDENLHCVPVETANVTMQLLKIRSGDRPS YVELTFSQHVRCECRPLREKMKPERCGDAVGP GQIVGGVYLL The first four amino acid residues are unrelated amino acids (MRGS); the last ten amino acids are the 10 G epitope (C terminal tag); the two amino acids preceding the 10 G epitope are also unrelated amino acids (Gly-Pro); the 126 amino acid sequence between the unrelated amino acids (i.e. beginning after MRGS and preceding GP) is the PlGF sequence identical to amino acid positions 4 to 129 in SEQ ID NO:1.

P126(AA): P1GF mutant#1: SEQ ID NO: 7: MRGSAVPPQQWALSAGNGSSEVEVVPFQEVWGRSYCRALERLVDVVSEYP SEVEHMFSPSCVSLLRCTGCCG AA NLHCVPVETANVTMQLLKIRSGDRPS YVELTFSQHVRCECRPLREKMKPERCGDAVGP GQIVGGVYLL same as P126(DE) except the underlined amino acids (AA) are the two mutated amino acids

P126(GAA): P1GF mutant#2: SEQ ID NO: 8: MRGSAVPPQQWALSAGNGSSEVEVVPFQEVWGRSYCRALERLVDVVSEYP SEVEHMFSPSCVSLLRCTGC G G AA NLHCVPVETANVTMQLLKIRSGDRPS YVELTFSQHVRCECRPLREKMKPERCGDAVGP GQIVGGVYLL same as PlGF mutant #1 P126(AA) except an additional mutation at the amino acid two before AA is mutated from C to G

All three recombinant proteins were expressed in bacteria and all form insoluble inclusion bodies. After sonication, washing with 4 M urea in PBS and 2M urea in PBS, inclusion bodies were finally solubilized by 8M urea/15 mM reduced Glutathione (GSH)/50 mM Tris-HCL (pH7.8). PlGF proteins were refolded through three step dialysis: (1) 24 hours against dialysis buffer 3M urea/50 mM TRIS(pH7.5)/2 mM EDTA/0.2 M Arginine/2 mM GSH, (2) 24 hours against dialysis buffer 2M urea/50 mM TRIS(pH7.5)/2 mM EDTA/0.2 M Arginine/1.2 mM GSH/0.4 mM oxidized Glutathione (GSSG) and (3) 24 hours against dialysis buffer 0.8M urea/20 mM TRIS(pH7.5)/2 mM EDTA/0.2 M Arginine/0.48 mM GSH/0.16 mM GSSG. Refolded PlGF were further purified by loading dialyzed protein solution to an affinity column, prepared by cross linking monoclonal antibody specific to 10 G tag and CNBr-activated Sepharose 4 Fast Flow resin (GE catalogue #17-0981-01). The bound PlGF was then eluted by 40% acetonitrile. Purified PlGF proteins were finally obtained after buffer exchange to PBS.

(2) Monoclonal anti-human PlGF, monoclonal anti-human sFlt are available from R&D Systems (Minneapolis, Minn. USA); HRP conjugated donkey anti-rat IgG (Cat #712-035-150) are available from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pa., USA); ELISA plates were Costar hind binding by Corning Life Sciences (Cat #2592); Electrophoresis gels NuPAGE 4-12%, PVDF transfer membrane and SeeBlue ladder were from Invitrogen (Carlsbad Calif., USA); Blocker casein/PBS and SuperSignal West Dura western blot substrate were purchased from Pierce (Rockford, Ill., USA). CNBr-activated Sepharose 4 Fast Flow resin (Cat #17-0981-01) and Silver stain kit (Cat #17-1150-01) were from GE Healthcare (Piscataway, N.J., USA)

Experiment Examples

(1) Recombinant PlGF: FIG. 2 shows the purity of the three PlGF recombinant proteins by silver stain and western blot.

(2) ELISA Assay-1

-   -   High-binding microtiter plate was coated with recombinant         P126(DE) or P126(AA) or P126(GAA) at 0.5 ug/mL and blocked with         BSA/PBS     -   Standard ELISA procedure consists of monoclonal antibody         dilution in casein/PBS; dilution of HRP conjugated donkey         anti-mouse IgG or donkey anti-rat IgG at 1:3 K in casein/PBS;         100 uL/well sample or conjugate volume; each step incubation at         37 C/30 min/shake; 6 times plate washing, 100 uL OPD substrate         development for 25 C/30 min; 25 uL stop solution; record OD at         492 nm.     -   ELISA results are shown in Table 4.

TABLE 4 Recognition of monoclnal anti-PlGF to recombinant PlGF (unaltered and altered) Antibody binding activity to coated PlGF (OD) Monoclonal anti-PlGF ID, clone # and Coated concentration (ng/mL) recombinant Ms-1 Rat 1 Rat 2 Rat 4 Rat 3 Rat 5 PlGF Ab at 1 ng/mL Ab at 10 ng/mL (0.5 ug/mL) 37203 358903 358939 358905 358932 358907 P126 (DE) 1.023 1.368 1.497 1.634 0.779 0.834 P126 (AA) 0.967 1.301 1.633 1.681 0.681 0.697 P126 (GAA) 1.134 1.226 1.530 1.591 0.806 0.799

-   -   Conclusion: All monoclonal antibodies tested reacted to         P126(DE), P126(AA) and P126(GAA), indicating that D72A/E73A         double mutation and C70G/D72A/E73A triple mutation did not         affect monoclonal antibody binding and these antibody epitope         locations were not at these mutation sites.         (3) ELISA Assay-2:     -   High-binding microtiter plate was coated with recombinant         P126(DE), P126(AA) and P126(GAA) at 0.5 ug/mL and blocked with         BSA/PBS     -   Standard ELISA procedure consists of 1^(st) plate incubation         with diluted sFlt in casein/PBS at various concentration, 2^(nd)         plate incubation with mixed anti-sFlt solution comprising two         monoclonal antibodies of RD-1 and RD-2 each at 0.1 ug/mL, 3rd         plate incubation with HRP conjugated donkey anti-mouse IgG at         1:4 K dilution in casein/PBS and 4^(th) plate incubation with         100 uL OPD substrate development for 25 C/30 min. 1^(st), 2^(nd)         and 3^(rd) plate incubation step is at 37 C/15-20 min/shake; 6         times plate washing between each step. 25 uL stop solution after         4^(th) incubation and record OD at 492 nm.     -   ELISA results are shown in Table 5.

TABLE 5 Binding of sFlt to recombinant PlGF (unaltered and altered) Complex formation of sFlt to coated PlGF sFlt concentration Coated recombinant (ng/mL) at incubation PlGF at (5 ug/mL) 1800 600 200 66.67 BSA (control) 0.009 0.008 0.005 0.003 P126 (DE) >3 1.833 0.833 0.347 P126 (AA) 0.210 0.182 0.115 0.143 P126 (GAA) 0.150 0.101 0.095 0.088 * Bound sFlt:PlGF complex were detected by mouse anti-sFlt and HRP anti-mouse conjugate

-   -   Conclusion: sFlt formed receptor:ligand complex with coated         P126(DE). However, such complex formation was greatly reduced         with P126(AA) mutant and P126(GAA) mutant, indicating that amino         acid position 70, 72 and 73 in SEQ ID NO:1 were critical for         sFlt-1 binding and complex formation.         (4) ELISA Assay-3:     -   High-binding microtiter plate was coated with recombinant sFlt         at 0.5 ug/mL and blocked with BSA/PBS     -   Standard ELISA procedure consists of 1^(st) plate incubation         with diluted P126(DE) or P126(AA) or P126(GAA) in casein/PBS at         various concentration, 2^(nd) plate incubation with monoclonal         anti-PlGF Rat-4 solution at 0.1 ug/mL, 3^(rd) plate incubation         with HRP conjugated donkey anti-Rat IgG at 1:4 K dilution in         casein/PBS and 4^(th) plate incubation with 100 uL OPD substrate         development for 25 C/30 min. 1^(st), 2^(nd) and 3^(rd) plate         incubation step is at 37 C/15-20 min/shake; 6 times plate         washing between each step. 25 uL stop solution after 4^(th)         incubation and record OD at 492 nm.     -   ELISA results are shown in Table 6.

TABLE 6 Binding of recombinant PlGF (unaltered and Complex formation of PlGF (unaltered or altered) to Coated PlGF (ng/mL) at sFlt at (0.5 1000 500 250 100 0 P126 (DE >3 1.388 0.557 0.259 0.021 P126 (A 0.299 0.118 0.101 0.077 0.009 P126 (GA 0.119 0.117 0.069 0.088 0.051 * Bound sFlt:PlGF complex were detected by mouse anti-PlGF (Rat-4) and conjugat

-   -   Conclusion: unaltered PlGF, P126(DE), formed ligand:receptor         complex with coated sFlt. However, altered PlGF (P126(AA) and         P126(GAA) failed to form such complex, indicating that amino         acid position 70, 72 and 73 in SEQ ID No:1 were critical for         sFlt binding and complex formation.

The description of the specific embodiments of the invention is presented for the purposes of illustration. It is not intended to be exhaustive or to limit the scope of the invention to the specific forms described herein. It will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and scope of the invention as set forth in the claims.

All patents, patent applications, and publications cited herein are hereby incorporated by reference. 

We claim:
 1. A method of calibrating or running controls in an assay for sFlt-1 and PlGF, the method comprising: selecting as a calibrator or control a composition comprising known amounts of non-immobilized sFlt-1 and non-immobilized PlGF, wherein one or more amino acids or one or more non-amino acid groups of the non-immobilized sFlt-1 and/or the non-immobilized PlGF have been deleted, modified, or replaced with a different amino acid or non-amino acid group or groups thereby reducing or substantially eliminating formation of a non-covalent association complex between the non-immobilized sFlt-1 and the non-immobilized PlGF; contacting the composition with a first immobilized receptor specific for sFlt-1 and a second immobilized receptor specific for PlGF, thereby forming a first complex of the non-immobilized sFlt-1 and the first immobilized receptor, and a second complex of the non-immobilized PlGF and the second immobilized receptor; and detecting amounts of the first complex and the second complex, wherein amounts of the first complex and the second complex are compared to the known amounts of the non-immobilized sFlt-1 and the non-immobilized PlGF to calibrate or control the assay.
 2. The method of claim 1 wherein the non-immobilized sFlt-1 is labeled with a first detectable label, and the non-immobilized PlGF is labeled with a second detectable label, and the detecting comprises detecting the first detectable label and the second detectable label.
 3. The method of claim 1 wherein the detecting comprises contacting the first complex with a first labeled moiety and the second complex with a second labeled moiety, thereby forming a labeled first complex and a labeled second complex, and wherein the detecting comprises detecting the labeled first complex and the labeled second complex.
 4. The method of claim 1 wherein the PlGF is PlGF-1.
 5. The method of claim 4 wherein PlGF-1 comprises alanine in place of: a) proline at position 25 of SEQ ID NO: 1, or b) glutamine at position 27 of SEQ ID NO: 1, or c) cysteine at position 60 of SEQ ID NO: 1, or d) aspartate at position 72 of SEQ ID NO: 1 or e) glutamate at position 73 of SEQ ID NO: 1, or f) asparagine at position 84 of SEQ ID NO: 1, or g) proline at position 98 of SEQ ID NO: 1, or h) tyrosine at position 100 of SEQ ID NO:1, or altered PlGF-1 having glycine in place of cysteine at position 70 of SEQ ID NO: 1, or any combination of the alanine replacements in a) to h) and the glycine replacement.
 6. The method of claim 4 wherein PlGF-1 comprises alanine in place of aspartate at position 72 of SEQ ID NO: 1 and alanine in place of glutamate at position 73 of SEQ ID NO:
 1. 7. The method of claim 4 wherein PlGF-1 comprises glycine in place of cysteine at position 70 of SEQ ID NO: 1, alanine in place of aspartate at position 72 of SEQ ID NO: 1 and alanine in place of glutamate at position 73 of SEQ ID NO:
 1. 